Pirin does not bind to p65 or regulate NFκB-dependent gene expression but does modulate cellular quercetin levels.

Pirin is a nonheme iron-binding protein with a variety of proposed functions, including serving as a coactivator of p65 NFκB and quercetinase activity. We report here, failure to confirm pirin's primary proposed mechanism, binding of Fe(III)-pirin and p65. Analytical size exclusion chromatography and fluorescence polarization studies did not detect an interaction.

Pirin does not bind to p65 or regulate NFκB-dependent gene expression but does modulate cellular quercetin levels.

Pirin is a non-heme iron binding protein with a variety of proposed functions including serving as a co-activator of p65 NFκB and quercetinase activity. We report here, failure to confirm pirin's primary proposed mechanism, binding of Fe(III)-pirin and p65. Analytical size exclusion chromatography (SEC) and fluorescence polarization (FP) studies did not detect an interaction.

Development of a Cell-Based AlphaLISA Assay for High-Throughput Screening for Small Molecule Proteasome Modulators.

The balance between protein degradation and protein synthesis is a highly choreographed process generally called proteostasis. Most intracellular protein degradation occurs through the ubiquitin-proteasome system (UPS). This degradation takes place through either a ubiquitin-dependent or a ubiquitin-independent proteasomal pathway.

Mass Spectrometry, Structural Analysis, and Anti-Inflammatory Properties of Photo-Cross-Linked Human Albumin Hydrogels.

Albumin-based hydrogels offer unique benefits such as biodegradability and high binding affinity to various biomolecules, which make them suitable candidates for biomedical applications. Here, we report a non-immunogenic photocurable human serum-based (HSA) hydrogel synthesized by methacryloylation of human serum albumin by methacrylic anhydride (MAA). We used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, liquid chromatography-tandem mass spectrometry, as well as size exclusion chromatography to evaluate the extent of modification, hydrolytic and enzymatic degradation of methacrylated albumin macromer and its cross-linked hydrogels.

Transforming Growth Factor 1 Increases Expression of Contractile Genes in Human Pulmonary Arterial Smooth Muscle Cells by Potentiating Sphingosine-1-Phosphate Signaling.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure and carries a very poor prognosis. Understanding of PAH pathogenesis is needed to support the development of new therapeutic strategies. Transforming growth factor (TGF-) drives vascular remodeling and increases vascular resistance by regulating differentiation and proliferation of smooth muscle cells (SMCs).

Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Antifibrotic and Antimetastatic Compounds.

A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF-dependent luciferase screen has shown remarkable efficacy in a variety of and models, including significant reduction of melanoma metastasis and bleomycin- induced fibrosis. Although these compounds are efficacious in these disease models, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent cotranscription factor, as a target of this series of compounds.

The Rho/MRTF pathway inhibitor CCG-222740 reduces stellate cell activation and modulates immune cell populations in Kras; Pdx1-Cre (KC) mice.

The stromal reaction in pancreatic cancer creates a physical barrier that blocks therapeutic intervention and creates an immunosuppressive tumor microenvironment. The Rho/myocardin-related transcription factor (MRTF) pathway is implicated in the hyper-activation of fibroblasts in fibrotic diseases and the activation of pancreatic stellate cells. In this study we use CCG-222740, a small molecule, designed as a Rho/MRTF pathway inhibitor.

5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent New Class of Inhibitors of Rho/Myocardin-Related Transcription Factor (MRTF)/Serum Response Factor (SRF)-Mediated Gene Transcription as Potential Antifibrotic Agents for Scleroderma.

Through a phenotypic high-throughput screen using a serum response element luciferase promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid lead inhibitor of Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated gene transcription with good potency (IC = 180 nM). We were able to rapidly improve the cellular potency by 5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable potency and depth of the SAR, as well as the relatively low molecular weight of the series, suggests, but does not prove, that binding to the unknown molecular target may be occurring through a covalent mechanism.

Local delivery of novel MRTF/SRF inhibitors prevents scar tissue formation in a preclinical model of fibrosis.

The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway represents a promising therapeutic target to prevent fibrosis. We have tested the effects of new pharmacological inhibitors of MRTF/SRF signalling in a preclinical model of fibrosis. CCG-222740, a novel MRTF/SRF inhibitor, markedly decreased SRF reporter gene activity and showed a greater inhibitory effect on MRTF/SRF target genes than the previously described MRTF-A inhibitor CCG-203971.

Pharmacokinetic optimitzation of CCG-203971: Novel inhibitors of the Rho/MRTF/SRF transcriptional pathway as potential antifibrotic therapeutics for systemic scleroderma.

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice.

Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis.

Small-Molecule Inhibition of Rho/MKL/SRF Transcription in Prostate Cancer Cells: Modulation of Cell Cycle, ER Stress, and Metastasis Gene Networks.

Metastasis is the major cause of cancer deaths and control of gene transcription has emerged as a critical contributing factor. RhoA- and RhoC-induced gene transcription via the actin-regulated transcriptional co-activator megakaryocytic leukemia (MKL) and serum response factor (SRF) drive metastasis in breast cancer and melanoma. We recently identified a compound, CCG-1423, which blocks Rho/MKL/SRF-mediated transcription and inhibits PC-3 prostate cancer cell invasion.

Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences.

Eph receptor signaling and ephrins.

The Eph receptors are the largest of the RTK families. Like other RTKs, they transduce signals from the cell exterior to the interior through ligand-induced activation of their kinase domain. However, the Eph receptors also have distinctive features.

Cancer somatic mutations disrupt functions of the EphA3 receptor tyrosine kinase through multiple mechanisms.

The Eph receptor tyrosine kinases make up an important family of signal transduction molecules that control many cellular processes, including cell adhesion and movement, cell shape, and cell growth. All of these are important aspects of cancer progression, but the relationship between Eph receptors and cancer is complex and not fully understood. Genetic screens of tumor specimens from cancer patients have revealed somatic mutations in many Eph receptors.